Enterotoxigenic E. coli (ETEC) is recognized as one of the leading causes of infectious diarrhea in developing countries (see, e.g., Black, Rev. Infect. Dis., 12 Suppl 1:S73-9 (1990)). The worldwide incidence of ETEC infections is estimated to result in 650 million cases of diarrhea and 380,000 deaths of children under the age of five (Gaastra et al., Trends Microbiol., 4(11): 444-52 (1996)). ETEC is also an important cause of traveler's diarrhea responsible for up to 60% of all cases among people traveling to endemic regions in Mexico (Adachi et al., J. Infect. Dis., 185(11):1681-3 (2002); Jiang et al., J. Infect. Dis., 185(4):497-502 (2002); Bouckenooghe et al., J. Travel. Med., 9(3):137-40 (2002)), Africa (Wolf et al., J. Clin. Microbiol., 31(4):851-6 (1993)), Bangladesh (Qadri et al., J. Clin. Microbiol., 38(1):27-31 (2000)), and Indonesia (Oyofo et al., Am. J. Trop. Med. Hyg, 65(2): 120-4 (2001)). ETEC is often linked to diarrheal outbreaks among American military personnel stationed abroad in Asia (Serichantalergs et al., J. Clin. Microbiol., 35(6): 1639-41(1997)) and the Middle East (Wolf et al., 1993, supra). While generally self-limiting and treatable with antibiotics, ETEC infection can negatively affect the quality of life for those afflicted, often resulting in the loss of several days before normal activities can be resumed. This is of particular concern to members of the military deployed to less developed countries and to vacationers traveling to countries endemic for ETEC.
The majority of ETEC cases are caused by the ingestion of bacterial enteropathogens in contaminated food or drink. Upon ingestion, ETEC colonize the upper intestinal tract facilitated by a variety of colonization factors. Once infection is established, ETEC secrete either a heat labile toxin (LT), a heat stable toxin (ST) or both. Although these toxins are antigenically distinct and bind to different host receptors, both elicit the production of a profuse, watery diarrhea. Additional symptoms may include intestinal cramps, nausea and, occasionally, more severe symptoms such as vomiting and fever. Among the 7.5 to 10 million cases of traveler's diarrhea reported annually, roughly 2 million are caused by LT-expressing ETEC (Steffen, R., Clin. Infect. Dis., 41 Suppl.(8):S536-40 (2005)).
ST is a small, monomeric toxin that contains multiple cysteine residues, whose disulfide bonds account for the heat stability of these toxins. There are two unrelated classes of STs that differ in structure and mechanism of action. Genes for both classes are found predominantly on plasmids, and some ST-encoding genes have been found on transposons.
LT is a prototypical A-B type toxin composed of an effector subunit (LT-A) and a cell-binding subunit (LT-B). LT-A is responsible for intoxication of host epithelial cells by activating host adenylate cyclase resulting in supraphysiological levels of cAMP. LT-B forms a pentameric complex that non-covalently interacts with LT-A to form LT holotoxin, which binds to the GM1 ganglioside associated with lipid rafts present on the host cell surface (Hirst, Cholera toxin and Escherichia coli heat-labile enterotoxin, in: The Comprehensive Sourcebook of Bacterial Protein Toxins, Alouf and Freer, eds. (San Diego, Academic Press, 1999), pp. 104-29). Once LT is bound to a host cell and endocytosed, LT-A is delivered to the cell cytoplasm where it exerts its toxic effects, leading to the extrusion of chloride, bicarbonate and water from the cell and net fluid loss from the intestine.
Table 1 below shows the incidence of LT and ST-expressing ETEC strains isolated from travelers to various geographical regions around the world.
TABLE 1Number of ETEC isolatedLocation#LT#LT-ST#ST% LT total*Egypt12252065%Saudi Arabia20595360%Egypt16174244%Jamaica2261074%Kenya30518349%India18332270%Mexico91535174%% LT total = LT only + LT/ST strains
As can be seen from Table 1, the incidence of disease from ETEC strains that express LT varies widely and is dependent on geographical location. Worldwide, roughly half of all ETEC strains produce LT (Wolf, Clin. Microbiol. Rev., 10(4):569-84 (1997)). Native populations in endemic areas are often most afflicted by ST-producing strains (Qadri et al., 2000, supra; Oyofo et al., 2001, supra). This may be because, even when infection rates of LT-ETEC are high, the number of symptomatic infections can be reduced by prior exposure (Steinsland et al., Lancet, 362(9380):286-91 (2003)), leading to the observation that the rate of asymptomatic infections by LT producing strains is typically greater than for ST producing strains (Qadri et al., Clin. Microbiol. Rev., 18(3):465-83 (2005)).
Travelers from the developed world to endemic regions develop ETEC infections of a different profile. In many areas of the world, LT-producing ETEC (LT only and LT-ST) are responsible for most cases of traveler's diarrhea (Bouckenooghe et al., 2002, supra; Wolf et al., 1993, supra; Estrada-Garcia, J. Travel. Med., 9(5):247-50 (2002); Rockabrand et al., Diagn. Microbiol. Infect. Dis., 55(1):9-12 (2006); Steffen et al., J. Travel. Med., 12(2):102-7 (2005)). It is clear from this distribution that a vaccine against LT-producing strains would protect travelers to many endemic areas against ETEC-related diarrhea.
The LTs of E. coli are oligomeric toxins which are closely related in structure and function to the cholera enterotoxin (CT) expressed by Vibrio cholerae (Sixma et al., J. Mol. Biol., 230:890-918 (1993)). Like LT, CT is composed of a single A subunit (CT-A) and a pentamer forming B subunit (CT-B) that binds GM1 ganglioside. LT and CT share many characteristics, including holotoxin structure, protein sequence (ca. 80% identity), primary receptor identity, enzymatic activity, and activity in animal and cell culture assays. LT and CT are additionally antigenically cross-reactive (Hirst, 1999, supra).
Attenuated Salmonella typhi has been used to deliver the B subunit of enterotoxinogenic E. coli (LT-B) (Stratford et al., Infect. Immun. 73(1):362-8 (2005)). The construct consisted of a single chromosomal copy of eltB expressed from the ssaG promoter. Mice inoculated subcutaneously or intranasally reportedly developed high titer responses to LT-B. In a Phase I clinical study, it was reported that 67% of vaccinees seroconverted after two doses (Khan et al., Vaccine, 25(21): 4175-82 (2007). It was noted, however, that in a similar construct, most of the LT-B was cell-associated, and release into the surrounding media was achieved only with cell lysis (Clements et al., Infect. Immun. 46(2):564-9 (1984)). Since the immunogenicity of LT-B and CT-B are associated with their ability to interact with host surface receptors (Nashar et al., Proc. Natl. Acad. Sci. USA, 93(1):226-30 (1996)), it is possible that a strain able to secrete LT-B or CT-B would be more effective for raising an anti-LT-B/anti-CT-B immune response. One object of the present invention is to discover whether this increased immunogenicity can be achieved.
Purified LT has also been tested as an ETEC vaccine. Although toxic when delivered orally and intranasally, transcutaneous delivery has shown potential. In a recent study, volunteers were vaccinated twice over a 21-day period by wearing an arm patch loaded with 50 μg of LT for 6-8 hours. All vaccinees reportedly seroconverted and developed a mean increase in serum IgG titers against LT of 24-fold (Glenn et al., Infect. Immun., 75(5):2163-70 (2007)). Neutralizing antibody against LT was also reportedly detected. In a subsequent study, subjects were vaccinated three times, on days 0, 21, and 42 by application of an arm patch, alternating arms with each visit (McKenzie et al., Vaccine, 25(18):3684-91 (2007)). Each subject then returned 18-26 hours later for patch removal. Subjects were then challenged with a wild-type ETEC strain. Although all of the vaccinees had seroconverted to LT, no protection against an LT/ST ETEC challenge was observed.
Orally delivered killed ETEC is another approach that has been attempted but has failed to show induction of protective immunogenicity: In a recent field study, an orally delivered killed vaccine was used, consisting of a combination of several ETEC strains, each expressing a different colonization factor, and 1 mg of CT-B per dose (Sack et al., Vaccine, 25(22):4392-400 (2007)). Travelers receiving two doses were monitored for several weeks after traveling to Mexico or Guatemala. No protection against vaccine-preventable diarrhea was observed, although the severity of disease was reportedly reduced in vaccinees.
Such studies indicate a possible utility for CT-B to promote cross-over immunity against LT/ETEC strains. However, they also dramatically illustrate the failure of previous vaccines to deliver CT-B by a route or in a form or quantity that would provide a useful level of anti-LT immunity.
Like ETEC, Vibrio cholerae, the causative agent of cholera, has long plagued mankind. There have been six pandemics of this disease caused by strains of V. cholerae belonging to the “Classical” biotype. The etiological agents of the current (seventh) pandemic belong to the “El Tor” biotype. Recently the seventh pandemic has extended to a new locale, that of South America. Beginning in January of 1991, an epidemic of cholera resulted in more than 250,000 cases and over 2,000 deaths in Peru, Ecuador, Columbia, and Chile. In November of 1992, an antigenically distinct, non-01 form of V. cholerae emerged in India and Bangladesh and within eight months caused an estimated 500,000 new cholera cases and 6,000 deaths. The pandemic potential of this new strain, designated serogroup 0139 synonym “Bengal”, seems assured and is a new cause of concern throughout the developing world.
Because natural infection by and recovery from cholera induces immunity lasting at least 3 years (Tacket et al., Cholera Vaccines, in Vaccines: New Approaches to Immunological Problems, Ellis, R. W., ed. (Butterworth-Heinenann, Boston, 1992); Levine et al., J. Infect. Dis., 143:818-820 (1981)), much effort has been made to produce a live, attenuated cholera vaccine that when administered orally would mimic the disease-producing wild type strains in its immunization properties but would not cause adverse symptoms or reactions in the immunized individual, i.e., display low reactogenicity. Attempts at developing vaccines of this type typically have involved deletion mutations that inactivate the gene encoding the A subunit of cholera toxin, a protein which is responsible for most of the diarrhea seen in this disease (Mekalanos et al., P.N.A.S. USA, 79:151-155 (1988); Mekalanos et al., Nature, 306:551-557 (1983); Kaper et al., Nature, 308:655-658 (1984); Kaper et al., Biotechnology, 2:345 (1984)). While both oral, killed whole cell vaccines and several live, attenuated cholera vaccines have been developed, the most promising of these provide little protection against the El Tor biotype of V. cholerae and probably no protection against the 0139 serotype.
V. cholerae only causes disease when colonization of the small bowel occurs. As a mucosal pathogen, V. cholerae adheres selectively to the M cells of the gastrointestinal tract (Owen et al., J. Infect. Dis., 153:1108-1118, (1986)) and is a strong stimulus to the mucosal immune system (Svennerholm et al., Lancet, 1:305-308, (1982)). This colonization of mucosa-associated lymphoid tissues (MALT) is also required for the induction of a localized immune response featuring locally produced secretory IgA, an important aspect of development of effective vaccines. (Holmgren & Czerkinsky, Nature Medicine Supp., 11(4):S45-S53 (2005).) Because of the importance of eliciting mucosal immunity in combating V. cholerae (and ETEC) infections, live avirulent vaccine strains are of particular interest: killed vaccines, although they might be highly immunogenic, do not have the ability to colonize gut or mucosa-associated tissues. A promising candidate for an effective live avirulent cholera vaccine is CholeraGarde® Peru-15 (under development by AVANT Immunotherapeutics, Inc., Needham, Mass.). Peru-15 is a live avirulent V. cholerae strain (genotype: ΔattRS1, Δcore ΔrecA:htpG:ctxB,Δctxφ) that has been shown to be well-tolerated and immunogenic in more than 400 subjects (U.S. Pat. No. 6,203,799; Kenner et al., J. Infect. Dis. 172:1126-1129 (1995); Cohen et al., Infect. Immun. 70: 1965-1970 (2002)).
An oral, killed, whole-cell cholera vaccine, Dukoral®, which includes the addition of 1 mg CT-B, was developed in the 1980s by SBL Vaccin of Sweden. While evaluating the efficacy of this vaccine against cholera in Bangladeshi women and children, it was discovered that the vaccine also provided short-term protection against LT/ST-producing ETEC and that protection was dependent on the inclusion of CT-B in the vaccine (Clemens et al., J. Infect. Dis., 158(2):372-7 (1988)).
In a later study, Finnish tourists traveling to Morocco received two doses of Dukoral® and were protected against disease caused by LT and LT/ST-expressing ETEC (Peltola et al., Lancet, 338(8778):1285-9 (1991)). Specifically, the WC/rBS cholera vaccine (Dukoral®) was reported to prevent 23% of all diarrhea episodes and 52% of episodes due to ETEC in Finnish tourists visiting Morocco. This protection was reported, however, not to last more than a few months. While the short-term protection was limited, these studies show CT-B as a possibly effective immunogen in the prevention of diarrhea against LT-producing ETEC in travelers.
As seen from the foregoing, many attempts have been made to address ETEC and V. cholerae infections and to provide protection against debilitating diseases for people living in or planning travel to endemic areas. However, the need remains for a composition that is effective to elicit an effective mucosal immune response to ETEC or V. cholerae, or ideally there is a great need for an immunogenic composition that would have the ability to raise a dual immune response, capable of eliciting neutralizing antibodies against both V. cholerae and ETEC pathogens. Potential vaccine strains providing dual immunogenicity in the form of a live, attenuated strain that could be administered orally and that retain the ability to colonize MALT tissues would be especially desirable.